Some members of Rhododendron genus are traditionally used as medicinal plants for arthritis, acute and chronic bronchitis, asthma, pain, inflammation, rheumatism, hypertension and metabolic diseases. To the best of our knowledge, there is no report on the protective effects of R. oldhamii leaf extract on non-alcoholic fatty liver disease (NAFLD) in vivo and in vitro.
Int J Med Sci 2017, Vol 14 Ivyspring International Publisher 862 International Journal of Medical Sciences 2017; 14(9): 862-870 doi: 10.7150/ijms.19553 Research Paper Rhododendron oldhamii leaf extract improves fatty liver syndrome by increasing lipid oxidation and decreasing the lipogenesis pathway in mice Ya-Ling Liu1*, Lei-Chen Lin2*, Yu-Tang Tung3, Shang-Tse Ho1, Yao-Li Chen4, Chi-Chen Lin5 and Jyh-Horng Wu1 Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan; Department of Forestry and Natural Resources, National Chiayi University, Chiayi 600, Taiwan; Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, Taipei 110, Taiwan; Division of General Surgery, Department of Surgery, Changhua Christian Hospital, Changhua 500, Taiwan; Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan * Equal contributions to this paper Corresponding author: Tel.: +886 22840345-136 Fax: +886 22851308 E-mail: eric@nchu.edu.tw (J.-H Wu); Tel.: +886 22840896-132 Fax: +886 22853469 E-mail: lincc@dragon.nchu.edu.tw (C.-C Lin) © Ivyspring International Publisher This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/) See http://ivyspring.com/terms for full terms and conditions Received: 2017.02.07; Accepted: 2017.05.21; Published: 2017.07.19 Abstract Some members of Rhododendron genus are traditionally used as medicinal plants for arthritis, acute and chronic bronchitis, asthma, pain, inflammation, rheumatism, hypertension and metabolic diseases To the best of our knowledge, there is no report on the protective effects of R oldhamii leaf extract on non-alcoholic fatty liver disease (NAFLD) in vivo and in vitro In this study, the effects of R oldhamii leaf extract on inhibiting the free fatty acid (FFA)-induced accumulation of fat in HepG2 cells and on improving fatty liver syndrome in mice with high fat diet (HFD)-induced NAFLD were investigated For the in vitro assay, HepG2 cells were treated with FFAs (oleate/palmitate = 2:1) with or without treatment with R oldhamii leaf ethyl acetate (EtOAc) fraction to observe lipid accumulation using Nile red and oil red O stains For the in vivo assay, C57BL/6 mice were randomly assigned to three groups (n = 5), including the normal diet group, the HFD group and the HFD+EtOAc group After 11 weeks, body weight, serum biochemical indices and the mRNA expressions of the liver tissue, as well as the outward appearance, weight and histopathological analysis of liver and adipose tissues were evaluated Among the fractions derived from R oldhamii leaf, the EtOAc fraction exhibited a strong fat-accumulation inhibitory activity Following reverse-phase high-performance liquid chromatography (HPLC), four specific phytochemicals, including (2R, 3R)-astilbin (AS), hyposide (HY), guaijaverin (GU) and quercitrin (QU), were isolated and identified from the EtOAc fraction of R oldhamii leaf extract Among them, AS and HY showed excellent fat-accumulation inhibitory activity Thus, the EtOAc fraction of R oldhamii leaf and its derived phytochemicals have great potential in preventing FFA-induced fat accumulation In addition, the EtOAc fraction of R oldhamii leaf significantly improved fatty liver syndrome and reduced total cholesterol (TC) and triglyceride (TG) in HFD-induced NAFLD mice at a dosage of 200 mg/kg BW These results demonstrated that the methanolic extracts from R oldhamii leaf have excellent inhibitory activities against fat accumulation and anti-NAFLD activities and thus have great potential as a natural health product Key words: Rhododendron oldhamii, free fatty acid (FFA), fat accumulation, high fat diet (HFD), non-alcoholic fatty liver disease (NAFLD) Introduction In 2014, an estimated 600 million adults were obesity according to the World Health Organization Obesity increases the risk of a number of health problems, including coronary disease, particular http://www.medsci.org Int J Med Sci 2017, Vol 14 types of carcinoma, respiratory system complications and osteoarthritis of small and large joints [1] Genetic, physiological and psychological factors, as well as dietary habits, physical activity, lifestyle and social and environmental factors are responsible for the significant increase in the prevalence of obesity and its consequences [1-4] Obesity is a condition when fat accumulation is excessive to the extent that it produces adverse health consequences [1] In the first stage of the two-hit hypothesis, fat accumulation in hepatocytes leads to steatosis, which is related to obesity [5] In addition, non-alcoholic fatty liver disease (NAFLD) has been considered the 2-stage process of the two-hit hypothesis The Rhododendron genus is widely distributed throughout most of the world except for Africa and South America [6] In traditional medicine, some members of the genus Rhododendron have been used to treat diseases, including arthritis, acute and chronic bronchitis, asthma, pain, inflammation, rheumatism, hypertension and metabolic diseases [7, 8] A variety of phytochemicals with significant bioactivities, including iridoids [9], diterpenoids [10], triterpenoids [11], chromane derivatives, [12] and flavonoids [13], have been discovered in this genus R groenlandicum is a popular beverage to treat diabetes symptoms [14] Ouchfoun et al [15] showed that R groenlandicum alleviates insulin resistance in a high fat diet (HFD)-induced obesity mice R arboreum has hypolipidemic activity in a diet-induced hypercholestermic rabbits [16, 17] In addition, the methanolic extract of R arboretum also showed significant in-vitro antidiabetic activity [18] Therefore, the present study was undertaken to investigate the 863 anti-NAFLD effect of R oldhamii leaf extract The anti-NAFLD effects of flavonoids in vitro and in vivo models have been reported in several studies [19] Flavonoids have been shown to help in treating and reducing the risk of obesity [20-22] In previous studies, plant catechins and anthocyanins reduced the weight of abdominal adipose tissues on diet induced obesity animal models [23] R oldhamii is rich in flavonoids, including (2R, 3R)-epicatechin, (2R, 3R)-taxifolin, (2R, 3R)-astilbin (AS), hyposide (HY), guaijaverin (GU) and quercitrin (QU) [24] Therefore, R oldhamii may be a good candidate for further development as a remedy for treating fatty liver syndrome However, to the best of our knowledge, there is no prior report on the improvement of R oldhamii leaf on fatty liver syndrome in HFD-induced NAFLD mice Thus, we used both lipid accumulation induced by free fatty acid (FFA; oleate/palmitate = 2:1) in HepG2 cells and HFD-induced NAFLD mouse model to investigate the anti-fatty liver effect of the methanolic extract from R oldhamii leaf Methods Plant materials The leaves of Rhododendron oldhamii Maxim were collected at the end of April 2011 from the Lion Head Mountain of Taipei county in Taiwan The species were confirmed by Dr Lei-Chen Lin of National Chiayi University Extraction, fractionation, and isolation Extraction, fractionation and isolation were followed by the method of Tung et al [24] The leaves were soaked in methanol at ambient temperature for Figure Chemical structures of four major phytocompounds isolated from the EtOAc fraction of R oldhamii leaf: (2R, 3R)-astilbin (AS), hyposide (HY), guaijaverin (GU) and quercitrin (QU) http://www.medsci.org Int J Med Sci 2017, Vol 14 days to obtain extract The crude extract was then fractionated with n-hexane, ethyl acetate (EtOAc), n-butanol (BuOH) and water to yield soluble hexane, EtOAc, BuOH and water fractions The major phytochemicals, AS, HY, GU and QU (Figure 1), from the EtOAc fraction were isolated and characterized by HPLC and NMR, respectively Cell culture The HepG2 cell line purchased from ATCC was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) The cells were incubated in a 5% CO2 incubator at 37°C Oil red O staining HepG2 cells (2 × 105 cells/well) were seeded into a 6-well plate incubated for 24 h to allow cell adherence First, mL of fresh medium containing test samples was added into the cultures After h of incubation at 37°C, 0.25 mM of FFAs (oleate/palmitate, 2:1) was added to the medium and incubated at 37°C for 24 h The cells were rinsed with cold phosphate buffered saline (PBS) and fixed in 1% paraformaldehyde for 30 After the cells were washed with 75% EtOH, the cells were stained for 20 in a mg/mL oil red O solution to determine hepatic lipid accumulation After the stain was removed, the cells were washed with PBS and then counterstained with hematoxylin for 20 s Representative photomicrographs (400× magnification) were conducted by a camera mounted onto a microscope Nile red staining Nile red staining was used to specifically stain the intracellular fat HepG2 cells (2 × 105 cells/well) were seeded into a 6-well plate and incubated for 24 h to allow cell adherence First, mL of fresh medium containing the test samples was added into the cultures After h of incubation at 37°C, mM of FFAs (oleate/palmitate, 2:1) was added to the medium and incubated at 37°C for 24 h The cells were collected using 0.05% Trypsin-EDTA and incubated with Nile red (1 μg/mL) in PBS for 10 After PBS washed, the cells were suspended in 1% formaldehyde and then measured by flow cytometry at a laser excitation wavelength of 488 nm Cell viability assay To measure the cytotoxicity on the test samples, HepG2 cells (1 × 104 cells/well) were seeded into a 24-well plate and incubated for 24 h to allow cell adherence First, mL of fresh medium containing the test samples was added into the cultures and incubated at 37°C for 24 h Following the removal of 864 the medium, mL of tetrazolium salt solutions (1 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in 10 mL DMEM) was added After h of incubation at 37°C, the medium was removed and 600 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals Absorbance was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader Animals The C57BL/6 mice (6 weeks old) were given a standard laboratory diet and distilled water ad libitum and kept on a 12 h light/dark cycle at 24 ± 2°C This study was conducted according to institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University and conformed to the guidelines of the protocol IACUC-10393 approved by the IACUC ethics committee After week of acclimatization, 15 mice were randomly divided into two groups: the normal group (n = 5) was fed a standard chow diet (ND) and the experimental group (n = 10) was fed a HFD The experimental mice were divided into two groups (n = 5/group): 1) HFD receiving no treatment (HFD) and 2) HFD receiving 50 mg/kg of the EtOAc fraction from R oldhamii leaf Food intake and body weight were recorded Following 11 weeks of treatment, mice were sacrificed at 18 weeks of age At the end of the experiment, each mouse was anesthetized, and the liver and epididymal fat pad tissues were collected Biochemical analysis of serum samples Mouse blood samples were centrifuged at 1,400 g at 4°C for 15 min, and the levels of serum glucose, glutamate-pyruvate transaminase (GPT), triglyceride (TG) and total cholesterol (TC) were measured using an autoanalyzer (Hitachi 7060, Hitachi, Japan) Pathological histology Liver and epididymal fat pad tissues were fixed in 10% buffered formaldehyde, and histologically examined with hematoxylin and eosin (H&E) staining RT-PCR Total RNA of the liver tissue was extracted using Trizol reagent (Invitrogen) following the protocol specified by the manufacturer RT-PCR was followed by the method of Tung et al [25] The gene expressions of genes (SREBP1, ACC, FAS, CPT1α, PPARα and PPARγ) using complementary DNA from liver tissue was analyzed GAPDH was used as an internal control http://www.medsci.org Int J Med Sci 2017, Vol 14 865 Figure Effects of R oldhamii leaf soluble fractions on intracellular lipid accumulation in HepG2 cells HepG2 cells were pretreated with 200 μg/kg of R oldhamii leaf crude and soluble fractions of hexane, EtOAc, BuOH and water in the presence of 0.25 mM of FFAs (oleate/palmitate, 2:1) for 24 h The cells were stained with Nile red and analyzed by flow cytometry, after which the percentage of lipid accumulation was quantitated (A) Cells were stained with oil red O and analyzed by spectrophotometry (B) Cytotoxicity of R oldhamii leaf soluble fractions in HepG2 cells was measured using the MTT assay (C) The results are presented as the mean ± SD (n = 3) The bars marked by different letters are significantly different at the level of p